The ubiquitin-specific protease 16s are a family of enzymes that cleave ubiquitin from ubiquitinated protein substrates, and are important in many cellular processes. Ubiquitin is a highly conserved polypeptide found in all eukaryotes, and its major function is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome. The ubiquitin-dependent proteolyic pathway is mediated by a diverse array of enzymes and is one of the major routes by which intracellular proteins are selectively destroyed. (See, e.g., Hochstrasser M., Current Opinion In Cell Biology 4:1024-1031 (1992)).
In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. As a part of this process, dubiquitinating enzymes disassemble ubiquitin polymers or ubiquiting-substrate conjugates. For example, the dubiquitinating enzyme, UbpA, is required for development of Dictoyostelium. More particularly, specific developmental transitions in Dictyostelium require degradation of specific proteins that require the disassembly of polyubiquitin chains by UbpA. (See, e.g., Lindsey et al., Journal of Biological Chemistry 273:29178 (1998)).
Deubiquitinating enzymes serve a number of functions in the ubiquitin-dependent proteolytic pathway. (See, e.g., Hochstrasser (1992), supra; Rose, I. A., In Current Communications In Molecular Biology: The Ubiquitin System, Schlesinger and Hershko (eds.) Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1988)). First, the enzymes cleave ubiquitin from biosynthetic precursors occurring either as a series of ubiquitin monomers in head-to-tail linkage or as fusions to certain ribosomal proteins (See, e.g., Finley and Chau, Annual Review of Cell Biology 7:25-69 (1991)). Secondly, ubiquitin is recycled from intracellular conjugates, both to maintain adequate pools of free ubiquitin, and to reverse the modification of inappropriately targeted proteins. Lastly, deubiquitinating reactions are important to the degradation of ubiquitinated proteins by the 26S proteasome, a complex ATP-dependent enzyme.
The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.
The present invention provides transgenic cells comprising a disruption in a ubiquitin-specific protease 16 gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of a ubiquitin-specific protease 16 gene. In another embodiment, the transgenic cells are derived from the transgenic animals described below. The cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the ubiquitin-specific protease 16 gene. The targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct. The targeting construct may also comprise other regulatory elements that may enhance homologous recombination.
The present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a ubiquitin-specific protease 16 gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the ubiquitin-specific protease 16 gene. In one aspect, the transgenic animals of the present invention are defective in the function of the ubiquitin-specific protease 16 gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a ubiquitin-specific protease 16 gene.
The present invention also provides methods of identifying agents capable of affecting a phenotype of a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a xe2x80x9cnormalxe2x80x9d or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods. The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the ubiquitin-specific protease 16 gene.
The present invention further provides a method of identifying agents having an effect on a ubiquitin-specific protease 16 expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control. Compounds that may have an effect on a ubiquitin-specific protease 16 expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The invention also provides cell lines comprising nucleic acid sequences of a ubiquitin-specific protease 16 gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the ubiquitin-specific protease 16 gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the ubiquitin-specific protease 16 gene, comprising the steps of contacting the ubiquitin-specific protease 16 gene with an agent and detecting an agent/a ubiquitin-specific protease 16 gene complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions associated with a disruption in a ubiquitin-specific protease 16 gene, and more particularly, to a disruption in the expression or function of the ubiquitin-specific protease 16 gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a ubiquitin-specific protease 16 gene""s expression or function, including administering to a subject in need, a therapeutic agent which effects a ubiquitin-specific protease 16 expression or function.
The present invention further provides methods of treating diseases or conditions associated with disrupted targeted gene expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated ubiquitin-specific protease 16 genes.
Definitions
The term xe2x80x9cgenexe2x80x9d refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.
The terms xe2x80x9cpolynucleotidexe2x80x9d and xe2x80x9cnucleic acid moleculexe2x80x9d are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term xe2x80x9cpolynucleotidexe2x80x9d includes single-, double-stranded and triple helical molecules.
xe2x80x9cOligonucleotidexe2x80x9d refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, usually single-stranded, that provides a 3xe2x80x2-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
A xe2x80x9cfragmentxe2x80x9d of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
The term xe2x80x9cgene targetingxe2x80x9d refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.
The term xe2x80x9chomologous recombinationxe2x80x9d refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.
The term xe2x80x9chomologousxe2x80x9d as used herein denotes a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be determined using a xe2x80x9cBLASTNxe2x80x9d algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
The term xe2x80x9ctarget genexe2x80x9d (alternatively referred to as xe2x80x9ctarget gene sequencexe2x80x9d or xe2x80x9ctarget DNA sequencexe2x80x9d or xe2x80x9ctarget sequencexe2x80x9d) refers to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination. The target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes. The target gene comprises a portion of a particular gene or genetic locus in the individual""s genomic DNA. As provided herein, the target gene of the present invention is a ubiquitin-specific protease 16 gene. A xe2x80x9cubiquitin-specific protease gene 16xe2x80x9d refers to a sequence comprising SEQ ID NO:1 or comprising the sequence encoding the target identified in Genebank as Accession No.: AA170316; GI NO1748849. In one aspect, the coding sequence of the target gene comprises SEQ ID NO:1 or comprises the target gene identified in Genebank as Accession No.: AA170316; GI:1748849.
xe2x80x9cDisruptionxe2x80x9d of a ubiquitin-specific protease 16 gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes which may be of animal, plant, prokaryotic, or viral origin. Disruption, for example, can alter or replace a promoter, enhancer, or splice site of a ubiquitin-specific protease 16 gene, and can alter the normal gene product by inhibiting its production partially or completely or by enhancing the normal gene product""s activity.
The term, xe2x80x9ctransgenic cellxe2x80x9d, refers to a cell containing within its genome a ubiquitin-specific protease 16 gene that has been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting.
The term xe2x80x9ctransgenic animalxe2x80x9d refers to an animal that contains within its genome a specific gene that has been disrupted by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).
As used herein, the terms xe2x80x9cselectable markerxe2x80x9d or xe2x80x9cpositive selection markerxe2x80x9d refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neor) gene are resistant to the compound G418. Cells that do not carry the Neor gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
A xe2x80x9chost cellxe2x80x9d includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention.
The term xe2x80x9cmodulatesxe2x80x9d as used herein refers to the inhibition, reduction, increase or enhancement of a ubiquitin-specific protease 16 function, expression, activity, or alternatively a phenotype associated with a disruption in a ubiquitin-specific protease 16 gene.
The term xe2x80x9camelioratesxe2x80x9d refers to a decreasing, reducing or eliminating of a condition, disease, disorder, or phenotype, including an abnormality or symptom associated with a disruption in a ubiquitin-specific protease 16 gene.
The term xe2x80x9cabnormalityxe2x80x9d refers to any disease, disorder, condition, or phenotype in which a disruption of a ubiquitin-specific protease 16 gene is implicated, including pathological conditions.